Citation: Skoková Habuštová, O.; Svobodová, Z.; Kodrík, D.; Sehnal, F. Cry3Aa Toxin Is Not Suitable to Control Lepidopteran Pest Spodoptera littoralis (Boisd.). Plants 2022, 11, 1312. https://doi.org/10.3390/ plants11101312 Academic Editors: Wei Wei and C. Neal Stewart, Jr. Received: 19 April 2022 Accepted: 13 May 2022 Published: 15 May 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). plants Article Cry3Aa Toxin Is Not Suitable to Control Lepidopteran Pest Spodoptera littoralis (Boisd.) Oxana Skoková Habuštová 1, * , Zde ˇ nka Svobodová 1 , Dalibor Kodrík 1,2 and František Sehnal 1,† 1 Institute of Entomology, Biology Centre, Czech Academy of Sciences, 370 05 ˇ Ceské Budˇ ejovice, Czech Republic; svobodova@entu.cas.cz (Z.S.); kodrik@entu.cas.cz (D.K.) 2 Faculty of Science, University of South Bohemia, 370 05 ˇ Ceské Budˇ ejovice, Czech Republic * Correspondence: habustova@entu.cas.cz † Deceased author. Abstract: The toxicity of the Bacillus thuringiensis (Bt) toxin Cry3Aa—originally used against the main potato pest, the Colorado potato beetle, Leptinotarsa decemlineata—was verified on this species and then evaluated against the Egyptian armyworm, Spodoptera littoralis, which is a pest of several economically important plants. Larvae of S. littoralis were fed a semi-artificial diet supplemented either with a recombinant or with a natural Bt toxin Cry3Aa and with the genetically engineered (GE) potato of variety Superior NewLeaf (SNL) expressing Cry3Aa. Cry3Aa concentration in the diet and the content in the leaves were verified via ELISA (enzyme-linked immunosorbent assay) and HPLC (high-performance liquid chromatography) during and at the end of the experiments. The biological effectiveness of the coleopteran-specific Cry3Aa with previous reports of activity against S. littoralis was tested on five different populations of S. littoralis larvae by monitoring 13 parameters involving development from penultimate instar, weight, the efficiency of food conversion to biomass, ability to reproduce, and mortality. Although some occasional differences occurred between the Cry3Aa treatments and control, any key deleterious effects on S. littoralis in this study were not confirmed. We concluded that the Cry3Aa toxin appears to be non-toxic to S. littoralis, and its practical application against this pest is unsuitable. Keywords: Spodoptera littoralis; Leptinotarsa decemlineata; recombinant Cry3Aa; natural Cry3Aa; Superior NewLeaf; integrated pest management; biological control 1. Introduction One of the environmentally friendly methods used to reduce insect pest populations is the practical utilisation of the insecticidal crystal protein (Cry) that occurs naturally in the soil bacterium Bacillus thuringiensis (Ber.) (Bt). Cry toxins are usually applied via spraying or in genetically engineered (GE) plants. Cry toxins kill host cells and thus allow Bt germination in dead arthropods. Cry toxins are intestinal pore-forming δ-endotoxins that, after activation by host proteases in the midgut, interact with receptors on the midgut epithelium. For example, in Lepidoptera, aminopeptidase N (APN) receptors, cadherin-like receptors, and ATP binding cassette (ABC) protein family function as toxin receptors for Cry1A. They are involved in the cleavage of the amino-terminal end, including the helix, and the formation of a pre-pore oligomer of Cry toxin, which leads to membrane insertion and pore formation. The pore formation results in osmotic cell lysis or else activation of the oncotic cell death pathway [1,2]. Because of their interaction with greatly diversified receptors, Cry toxins are highly specific to certain species of the insect orders Lepidoptera, Coleoptera, Hymenoptera, Diptera, Orthoptera, and Mallophaga, and also to Nematoda, Acari, and Protozoa [3]. However, some Cry toxins have an expanded spectrum of action to two or more taxonomic categories. For example, Cry1B is one of those that present a remarkable activity against larvae of Lepidoptera, Diptera, and Coleoptera [4]. Plants 2022, 11, 1312. https://doi.org/10.3390/plants11101312 https://www.mdpi.com/journal/plants